PROJECT SUMMARY Recent evidence suggests that tumors may exploit immune inhibitory mechanisms to create a barrier against antitumor immune responses ? including endogenous inflammatory immune responses and those induced by immunotherapies. One key mechanism appears to be mediated by the programmed death-1 (PD-1)/B7 homolog 1 (B7-H1) pathway, which restrains antitumor T cell function in the tumor microenvironment (TME). PD-1 is induced on the surface of activated T cells and, after engaging its ligand, down-modulates T cell functions. Our early studies include cloning and identification of B7-H1 (PD-L1, CD274) as an inhibitory ligand for T cell response and characterization of B7-H1 as a major ligand for PD-1 in suppressing tumor immunity in TME. We demonstrated that B7-H1 was upregulated on the cell surface by inflammation (mainly via IFN-gamma), and that high levels of B7-H1 expression were found in many human cancers including non-small cell lung cancer (NSCLC). Blocking the interaction of B7-H1 on tumor cells with PD-1 on tumor-specific T cells can eliminate this barrier in the TME and protect ongoing antitumor immunity, leading to tumor regression in mouse tumor models. Recent clinical trials have demonstrated that anti-PD-1 or anti-B7-H1 monoclonal antibodies (mAb) induced objective clinical responses in a significant fraction of patients with advanced chemo-refractory metastatic NSCLC. Responses were highly durable with manageable toxicity. While these clinical findings are promising, it is now critical to better understand the effects of PD-1/B7-H1 blockade on antitumor immunity and to develop new strategies to overcome resistance. In this project, we will test the hypothesis that NSCLC is immunologically heterogeneous and only a subgroup of NSCLC with membranous B7-H1 (PD-L1) expression on cancer cells and the presence of functional tumor-infiltrating lymphocytes (TILs) will respond to anti-PD- 1/B7-H1 therapy, whereas resistance to anti-PD-1/B7-H1 therapy is largely mediated via PD-1/B7-H1- independent suppressive pathways. We will identify subgroups of NSCLC patients who respond to and resist anti-PD-1/B7-H1 therapy by analysis of B7-H1 expression and immune responses in the TME. In addition, we will delineate cellular and molecular mechanisms underlying resistance to anti-PD-1/B7-H1 therapy in each NSCLC subgroup. Finally, we will maximize the therapeutic effect of PD-1/B7-H1 blockade by additionally targeting resistance mechanisms using mouse models with induced lung cancer. The current proposal integrates basic and clinical sciences, and will use animal models and human specimens in the context of ongoing clinical trials of anti-PD-1 and B7-H1 antibodies to achieve the goals. Taken together, results from our studies will enhance future anti-PD-1/B7-H1 therapy and potentially lead to novel immune-based therapies for lung cancer.